This application is in response to Notice Number (NOT-OD-09-058) and Notice Title: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications. We propose a competitive revision to the Clinical Proteomic Technology Assessment for Cancer (CPTAC) program at Vanderbilt, which will support expanded studies of quantitative analysis methods for phosphotyrosine proteomes. Signaling pathways based on tyrosine phosphorylation are critical to the development of cancer and drugs that inhibit tyrosine kinases are effective therapeutic agents. Despite their widespread use, utility of tyrosine kinase inhibitors is limited by the emergence of resistance, apparently involving compensating signaling mechanisms. Proteomic approaches for global assessment of phosphotyrosine signaling are needed to investigate these mechanisms underlying resistance and response. Shotgun proteomics using liquid chromatography-tandem mass spectrometry combined with immunoaffinity enrichment of phosphotyrosine peptides enables global analysis, but lacks robust quantitative performance. Moreover, isotope labeling approaches (SILAC, iTRAQ) are not applicable to human tissue specimens and provide only relative quantitation in paired sample analyses. We propose to develop and evaluate a new label-free method for quantitative analysis of phosphotyrosine proteins. This approach uses tyrosine phosphorylated bacterial proteins to normalize mass spectral signals for endogenous phosphopeptide quantification in immunoaffinity shotgun analyses and for assessment of variability due to digestion, immunoprecipitation and other sample preparation steps. We will apply liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM-MS) analysis methods for sensitive, targeted quantification. We will validate this approach through analysis of HER2-regulated phosphotyrosine signaling in a human breast cancer cell model and in tumor tissue from a HER2-regulated mouse xenograft model. This new work will leverage existing collaborations and resources within the Vanderbilt CPTAC to facilitate implementation with new standards and data analysis tools. PUBLIC HEALTH RELEVANCE: Reliable methods to measure disease-related protein changes in cancer are needed to advance the field of cancer diagnostics. The proposed work will develop and test methods to reliably measure protein phosphorylation changes in response to cancer development and anticancer drug therapy.